A recent public announcement by the US National Institutes of Health states: "Biofilms are medically important, accounting for over 80 percent of microbial infections in the body". Bacterial adhesion to surfaces is also a major industrial problem, affecting water purification systems, heat exchangers, biological sensors and many others. However, in-depth understanding of biofilm formation and strategies to control its formation, remain lacking. Adaptation of bacterially-derived, biofilm-degrading enzymes offers a novel option to biofitm control. Bacitracin, a peptide antibiotic derived from the biofilm-forming Bacillus licheniformis, inhibits the cell wall formation of Gram-positive bacteria. In humans, the use of this antibiotic is limited by its toxicity. In animals, however, bacitracin is widely used as a growth-promoting food additive and thought to contribute to the development of antibiotic resistance. We have found commercial bacitracin, including bacitracin formulated for human use by injection, to be contaminated with significant quantities of bacterially-derived protease and DNAse. Our data show that these enzymes are exceptionally active, resilient, and show biofilm-degrading capabilities. This research will contribute to our understanding of the degradative enzymes found in antibiotic bacitracin and characterize their impact on biomedically-relevant biofilms. Future studies will aim to immobilize these degradative enzymes on diverse biomaterial surfaces and assess their impact on biofitm formation and antibiotic susceptibility. In our current proposal, we plan on addressing the following specific aims: AIM 1. Biochemical characterization, purifcation, sequencing, cloning, genetic analysis and bioinformatics of the bacitracin-contaminating protease and DNAse; AIM 2. Establishment of in vitro model-systems of medically-relevant biofilms, followed by an investigation of the impact of these degradative enzymns on biofilm removal, growth inhibition and enhancement of susceptibility to biocides and antibiotics. Future studies will focus on surface modification of medical biomaterials (e.g. methacrytates, hydroxyapatite, gold-coated surfaces) with these enzymes and subsequent evaluation of the biofilm-forming processes on modified materials. Other applications of these degradative enzymes for biotechnology applications will also be evaluated. This work will be carried out in collaboration with three other NMTech Biology Department faculty members. In the broader context, this interdisciplinary project will establish a new area of biomedical research at New Mexico Tech: biofilm research.